Acclimation of Chlamydomonas Reinhardtii to Nitric Oxide Stress Related To

Home , Cytochrome b6f complex, Photosystem, Photosystem I, Photosystem II

1 Short Title: Acclimation of Chlamydomonas to Sub-Lethal NO

3 Author for contact details

4 Tse-Min Lee

5 Department of Marine Biotechnology and Resources, National Sun Yat-sen University,

6 Kaohsiung 80424, Taiwan

7 e-mail: [email protected]

8 Acclimation of Chlamydomonas reinhardtii to Nitric Oxide Stress Related to

9 Respiratory Burst Oxidase-Like 2

11 Eva YuHua Kuo and Tse-Min Lee

12 Department of Marine Biotechnology and Resources, National Sun Yat-sen University,

13 Kaohsiung 80424, Taiwan

15 One-sentence Summary: Acclimation machinery is triggered in Chlamydomonas

16 reinhardtii cells against sub-lethal nitric oxide stress.

18 Footnotes

19 Author Contributions: E.Y.K. performed the experiments; T.M.L. analyzed the data

20 and wrote the article; all authors edited and approved the manuscript; T.M.L. is the

21 author responsible for contact and ensures communication.

22 Responsibilities of the Author for Contact: The author responsible for distribution

23 of materials integral to the findings presented in this article in accordance with the

24 policy described in the Instructions for Authors (www.plantphysiol.org) is Tse-Min

25 Lee ([email protected]).

27 Funding Information: This research was supported by grants from the Ministry of

28 Science Technology, Executive Yuan, Taiwan (MOST 103-2311-B-110-001MY3 and

29 MOST 107-2311-B-110-003-MY3) to TML.

31 Email Address of Author for Contact

32 Tse-Min Lee: [email protected]

33 Abstract

34 The acclimation mechanism of Chlamydomonas reinhardtii to nitric oxide (NO) was

35 studied by exposure to S-nitroso-N-acetylpenicillamine (SNAP), a NO donor.

36 Treatment with 0.1 or 0.3 mM SNAP transiently inhibited photosynthesis within 1 h,

37 followed by a recovery without growth impairment, while 1.0 mM SNAP treatment

38 caused irreversible photosynthesis inhibition and mortality. The SNAP effects are

39 avoided in the presence of the NO scavenger,

40 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO).

41 RNA-seq, qPCR, and biochemical analyses were conducted to decode the metabolic

42 shifts under sub-lethal NO stress by exposure to 0.3 mM SNAP in the presence or

43 absence of 0.4 mM cPTIO. These findings revealed that the acclimation to NO stress

44 comprises a temporally orchestrated implementation of metabolic processes: 1. trigger

45 of NO scavenging elements to reduce NO level; 2. prevention of photo-oxidative risk

46 through photosynthesis inhibition and antioxidant defense system induction; 3.

47 acclimation to nitrogen and sulfur shortage; 4. degradation of damaged proteins

48 through protein trafficking machinery (ubiquitin, SNARE, and autophagy) and

49 molecular chaperone system for dynamic regulation of protein homeostasis. NO

50 increased NADPH oxidase activity and respiratory burst oxidase-like 2 (RBOL2)

51 transcript abundance, which were not observed in the rbol2 insertion mutant. Changes

52 in gene expression in the rbol2 mutant and increased mortality under NO stress

53 demonstrate that NADPH oxidase (RBOL2) is involved in the modulation of some

54 acclimation processes (NO scavenging, antioxidant defense system, autophagy, and

55 heat shock proteins) for Chlamydomonas to cope with NO stress. Our findings provide

56 insight into the molecular events underlying acclimation mechanisms in

57 Chlamydomonas to sub-lethal NO stress.

59 INTRODUCTION

60 Nitric oxide (NO) is a crucial signaling molecule in diverse physiological processes

61 in plants (Durner et al., 1999), including in the green alga Chlamydomonas, in which

62 NO regulates many physiological processes and stress responses such as the

63 remodeling of chloroplast proteins by the degradation of thylakoid cytochrome b6f

64 complex and stroma ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) via

65 FtsH and Clp chloroplast proteases under nitrogen (Wei et al., 2014) or sulfur

66 starvation (de Mia et al., 2019). In Chlamydomonas, NO is also involved in cell death

67 induced by ethylene and mastoparan (Yordanova et al., 2010), induction of oxidative

68 stress under extreme high light (VHL, 3,000 μmol∙m-2∙s-1) (Chang et al., 2013),

69 interaction of NO with hydrogen peroxide (H2O2) for high light stress-induced

70 autophagy and cell death (Kuo et al., 2020a), proline biosynthesis under copper

71 stress (Zhang et al., 2008), and responses to salt stress (Chen et al., 2016). NO is

72 responsible for the regulation of nitrogen assimilation by repressing the expression of

73 nitrate reductase as well as nitrate and ammonium transporters (de Montaigu et al.,

74 2010) and their enzyme activities (Sanz-Luque et al., 2013; Calatrava et al., 2017).

75 Mitochondrial respiration by upregulation of alternative oxidase 1 is modulated by NO

76 in Chlamydomonas (Zalutskaya et al., 2017).

77 Information is still lacking on the comprehensive overview of the transcriptional

78 regulation of C. reinhardtii in response to NO stress. Therefore, in the present study, C.

79 reinhardtii cells were treated with different concentrations (0.1, 0.3, and 1.0 mM) of

80 the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in the presence or absence of

81 an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide

82 (cPTIO) (Mur et al., 2011). This study provides new insight into the acclimation

83 mechanisms against NO stress in Chlamydomonas. 4

84 85

86 RESULTS

87 Physiological and Transcriptomic Changes in Response to NO Burst

88 The mechanisms that allow for the acclimation of C. reinhardtrii cells to short-term

89 NO burst were examined. The SNAP concentration and treatment duration were

90 carefully chosen to allow for the monitoring of the short-term response to NO rather

91 than cell death. Using a cell permeable NO-sensitive fluorescent dye, the fluorescence

92 emitted from the cells (Fig. 1A) rapidly increased 0.5 h after SNAP treatment and

93 reached a plateau after 1 h (Fig. 1B); the increase in fluorescence can be inhibited in

94 the presence of 0.4 mM cPTIO. Both the 0.1 and 0.3 mM SNAP treatments did not

95 affect cell viability (Fig. 1C) and growth (Fig. 1D), whereas the 1.0 mM SNAP

96 treatment resulted in cell mortality and bleaching (Fig. 1E). The concentrations of

97 chlorophyll a (Supplemental Fig. S1A), chlorophyll b (Supplemental Fig. S1B), and

98 carotenoids (Supplemental Fig. S1C) were not influenced by either 0.1 or 0.3 mM

99 SNAP treatments; however, a decrease in chlorophyll a, chlorophyll b, and carotenoids

100 6 h after treatment with 1.0 mM SNAP was observed. Photosynthesis is relatively

101 sensitive to NO burst, as reflected by a transient decrease in Fv′/Fm′ (Fig. 2A), Fv/Fm

102 (Fig. 2B), and the O2 evolution rate (Fig. 2C) 0.5 h after SNAP treatment; these

103 photosynthesis-related factors then recovered after 3–5 h in both the 0.1 and 0.3 mM

104 SNAP treatments but not in the 1.0 mM SNAP treatment. This inhibition of

105 photosynthesis-related factors can be suppressed in the presence of 0.4 mM cPTIO.

106 Based on fluorescence induction kinetics (OJIP curve), the cells treated with 0.1 or 0.3

107 mM SNAP transiently lost both the PSII acceptor-side (FJ-Fo and FI-FJ) and

108 donor-side (FP-FI and Fv/Fo) electron transfer ability, whereas 1.0 mM SNAP

109 treatment caused irreversible inhibition, which was prevented in the presence of 0.4

110 mM cPTIO (Supplemental Table S1). The SNAP treatments decreased the production

.- 1 111 of superoxide anion radical (O2 ) and H2O2; the production of singlet oxygen ( O2) 6

112 was significantly increased by 1.0 mM SNAP treatment (Supplemental Fig. S1D-F).

113 Treatment with 1.0 mM SNAP also caused significant lipid peroxidation

114 (Supplemental Fig. S1G). Overall, these results demonstrate two stages of response in

115 C. reinhardtii cells in the sub-lethal NO challenge: I. NO stress together with the

116 significant NO burst caused a sharp decline in photosynthetic activity 1 h after SNAP

117 treatment and II. the recovery period. Therefore, Chlamydomonas cells treated with

118 0.3 mM SNAP for 1 h in the presence or absence of 0.4 mM cPTIO were used for

119 transcriptomic analyses.

120 The results of the transcriptomic analysis (Supplemental Tables S2 and S3) showed

121 that 1,012 significant DEGs were regulated by NO (1.2 log2FC, P-value of log2FC ≤

122 0.05) (Supplemental Table S4). Following analysis using the MapMan display mode,

123 cellular response overview (Supplemental Fig. S2), proteasome and autophagy

124 (Supplemental Fig. S3), photosynthesis (electron transport, Calvin cycle, and

125 photorespiration) (Supplemental Fig. S4), and the tetrapyrrole pathway (Supplemental

126 Fig. S5) were affected under NO stress. Using the Blast2GO suite, the analysis of all

127 the functional DEGs and unigenes identified 45 GO terms (score ≤ 0.05) that can be

128 assigned to 463 upregulated genes (45.75%), with 13 biological process, 30 molecular

129 function, and two cellular component GO terms (Supplemental Fig. S6A and Table

130 S5A). One hundred fifty-seven GO terms for 549 downregulated genes with 337

131 known function genes (54.25%) were classified into 35 GO terms belonging to

132 biological process, 112 to molecular function, and eight to cellular component

133 (Supplemental Fig. S6B and Table S5B). The genes associated with amino acid

134 catabolism, the ubiquitin-proteasome system, and the antioxidant defense system are

135 upregulated, while those related to transcriptional and translational regulation are

136 downregulated by NO burst (Supplemental Tables S4 and S5).

137 We also found that the genes associated with photosynthesis are downregulated by 7

138 NO burst (Supplemental Tables S7). NO decreased the transcript abundances of

139 lumenal PsbP-like protein (PSBP4, Cre03.g182551.t1.2), which is linked to the

140 stability of photosystem II complex assembly (Ido et al., 2014); Lhl2

141 (Cre08.g384650.t1.2) belonging to high light-induced protein (Teramoto et al., 2004),

142 Lhl3 (Cre03.g199535.t1.1); pre-apoplastocyanin (PCY1, Cre03.g182551.t1.2),

143 cytochrome c6A (CYC4, Cre16.g670950.t1.2) acting as electron transfer between

144 cytochrome b6f complex and photosystem I (Marcaida et al., 2006); CHLH

145 (Cre07.g325500.t1.1); CHLI1 (Cre06.g306300.t1.2); CHLI2 (Cre12.g510800.t1.2),

146 CHLD (Cre05.g242000.t1.2); and GENOMES UNCOUPLED 4 (GUN4,

147 Cre05.g246800.t1.2). In contrast, NO increased the transcript abundance of

148 chlorophyll a/b binding protein (LHCBM9, Cre06.g284200.t1.2), Lhl1

149 (Cre08.g384650.t1.2), and Lhl4 (Cre17.g740950.t1.2) (Fig. 3). For the Calvin cycle,

150 NO also decreased the transcript abundance of the Rubisco small subunit 1 (RBCS1,

151 Cre02.g120100.t1.2), ribulose phosphate-3-epimerase (RPE2, Cre02.g116450.t1.2),

152 phosphoglycerate kinase (PGK1, Cre11.g467770.t1.1), glyceraldehyde-3-phosphate

153 dehydrogenase (GAP4, Cre12.g556600.t1.2), triose phosphate isomerase (TPI1,

154 Cre01.g029300.t1.2), sugar bisphosphatase (SBP2, Cre17.g699600.t1.2), a

155 pentatrichopeptide repeat protein that stabilizes Rubisco large subunit mRNA (PPR2,

156 Cre06.g298300.t1.1), a small protein for phosphoribulokinase deactivation (Howard et

157 al., 2008) (CP12, Cre08.g380250.t1.2), Rubisco large (RMT1, Cre16.g661350. t1.2

158 and a similar one, Cre16.g649700.t1.1) and small (RMT2, Cre12.g524500.t1.2)

159 subunit N-methyltransferase, and phosphoglycolate phosphatase (PGP3,

160 Cre06.g271400.t1.1) in the photorespiration (Fig. 4). In contrast, the transcript

161 abundance of Rubisco activase-like protein (RCA2, Cre06.g298300.t1.1) was

162 increased by NO (Fig. 4H). The NO-induced changes in gene expression occurred 1 h

163 after NO exposure, recovered to the same levels as the control after 3 h, and the 8

164 changes at 1 h can be inhibited by the presence of cPTIO.

165

166 NO Decreases Nitrogen and Sulfur Availability

167 The transcript abundances of the ammonium transporters AMT3

168 (Cre06.g293051.t1.1) (Fig. 5A), AMT6 (Cre07.g355650.t1.1) (Fig. 5B), and AMT7

169 (Cre02.g111050.t1.1) (Fig. 5C), and the ammonium assimilation enzymes, glutamate

170 synthase (GSN1, Cre13.g592200.t1.2) (Fig. 5D) and glutamate synthetase (GLN1,

171 Cre02.g113200.t1.1) (Fig. 5E) decreased 1 h after NO treatment and then recovered,

172 while that of glutamate dehydrogenase (GDH1, Cre09.g388800.t1.2) increased after 1

173 h (Fig. 5F).

174 Cysteine desulfurase (CSD4, Cre12.g525650.t1.2), which moves the sulfur from the

175 cysteine to sulfur-containing recipients, showed an increase in transcriptional

176 expression in the NO treatment (Fig. 5G). Cysteine can be oxidized to 3-sulfinoalanine

177 (Chai et al., 2005) by cysteine dioxygenase (CDO), and the transcript abundances of

178 CDO1 (Cre03.g174400.t1.1) (Fig. 5H) and CDO2 (Cre03.g163950.t1.1) (Fig. 5I)

179 increased after NO treatment. In the NO treatment, the transcript abundance of taurine

180 dioxygenase (TAUD1, Cre13.g569400.t1.2), which degrades taurine to sulfite and

181 aminoacetaldehyde, increased (Fig. 5J) but that of aspartate aminotransferase (AST5,

182 Cre06.g278249.t1.1), which converts 3-sulfinoalanine to pyruvate, decreased (Fig. 5K).

183 Thus, cysteine is catabolized to taurine and then degraded to sulfite instead of pyruvate.

184 Then, sulfite is oxidized by sulfite oxidase (SUOX) to avoid toxicity due to

185 accumulation (Hansch et al., 2007). However, NO did not affect the expression of

186 SUOX (Cre04.g217929.t1.1) (Supplemental Table S3).

187 The expression of genes related to amino acid degradation was transiently

188 upregulated by NO, including L-allo-threonine aldolase (ATA2, Cre10.g423550.t1.1)

189 (Fig. 5L), the aromatic amino acid hydroxylase-related protein (AAH1, 9

190 Cre01.g029250.t1.2) (Fig. 5M), and the catabolism of branched-chain amino acids

191 (valine, leucine and isoleucine; 2-oxoisovalerate dehydrogenase E1 component alpha

192 (BCKDHA, Cre12.g539900.t1.1) (Fig. 5N) and beta (BCKDHB, Cre06.g311050.t1.2)

193 (Fig. 5O) (Supplemental Table S6). The presence of cPTIO inhibited the changes

194 induced by SNAP treatment.

195 NO increased the transcript abundance of periplasmic arylsulfatase (ARS3,

196 Cre10.g430200.t1.2), arylsulfatase (ARS7, Cre01.g011901.t1.1), plasma membrane

197 sodium/sulfate co-transporters (SLT1, Cre12.g502600.t1.2; SLT2,

198 Cre10.g445000.t1.2), the chloroplast sulfate binding protein as the component of

199 chloroplast transporter (SULP3, Cre06.g257000.t1.2), and SNRK2.2 (SAC3,

200 Cre12.g499500.t1.1), an Snf1-like serine/threonine kinase responsible for the

201 repression of expression of genes responsible for sulfur starvation (Davies et al., 1999;

202 Gonzalez-Ballester et al., 2008). NO decreased the transcript abundance of ARS5

203 (Cre10.g431800.t1.2), ARS11 (Cre04.g226550.t1.2), ARS13 (Cre05.g239750.t1.2),

204 ARS14 (Cre05.g239800.t2.1), and ARS16 (Cre06.g293800.t1.1) (Fig. 6). The

205 presence of cPTIO inhibited these changes in response to SNAP treatment.

206

207 NO Modulates Protein Homeostasis and Quality

208 Based on the identified GO terms, NO induces protein degradation through

209 ubiquitination and membrane trafficking (Supplemental Fig. S6). For genes involved

210 in the ubiquitin-proteasome system (Supplemental Table S8), the transcript

211 abundances of AAA+-type ATPase (Cre16.g650150.t1.3), which is responsible for the

212 opening of the gates for substrate into the axial entry ports of the proteases (Yedidi et

213 al., 2017); ubiquitin-protein ligase E2 (Cre07.g342506.t1.2); ubiquitin-protein ligase

214 E3 A (UBE3A; Cre08.g364550.t1.3); probable E3 ubiquitin-protein ligase HERC1

215 (Cre02.g099100.t1.3); and ubiquitin fusion degradation protein (Cre03.g179100.t1.2), 10

216 which is similar to ubiquitin fusion degradation protein 1, were increased by NO

217 treatment (Fig. 7). In contrast, the transcript abundances of ubiquitin-conjugating

218 enzyme E2I (UBC9, Cre01.g019450.t1.1), which exhibits the activity of small

219 ubiquitin-like modifier (SUMO) E2 conjugase (Wang et al., 2008), a central enzyme in

220 SUMO conjugation for interaction with E1 to accept SUMO in the formation of a

221 SUMO~UBC9 thioester bond (Pichler et al., 2017), and ubiquitin-conjugating enzyme

222 E2 (UBC3, Cre03.g167000.t1.2) (Fig. 7D) decreased in the NO treatment (Fig. 7).

223 These changes induced by the SNAP treatment were inhibited in the presence of

224 cPTIO.

225 We detected most of the genes encoding endocytosis (Supplemental Table S9A) and

226 SNAREs (Supplemental Table S9B; soluble N-ethylmaleimide-sensitive factor

227 attachment protein receptor), which mediate fusion events for autophagosome

228 biogenesis as the membrane trafficking pathways. NO increased the transcript

229 abundances of the vacuolar protein sorting (VPS) proteins, which sort receptors within

230 the endocytic pathway (Bishop and Woodman, 2001) including the subunits of the

231 ESCRT-I (endosomal sorting complex I required for transport) complex (i.e., VPS23

232 (Cre07.g351050.t1.2), VPS28 (Cre16.g678100.t1.2), and VPS37

233 (Cre08.g362550.t1.2)), the subunit of the ESCRT-III complex (VPS2A;

234 Cre04.g213450.t1.2), and RAB5 (Cre12.g517400.t1.2), a small rab-related GTPase

235 that regulates vesicle formation and membrane fusion. NO also increased the transcript

236 abundances of SYNTAXIN-8B-RELATED (Cre06.g293100.t1.2), a member of the

237 syntaxin family that is involved in protein trafficking from early to late endosomes via

238 vesicle fusion (Prekeris et al., 1999), and endosomal R-SNARE protein belonging to

239 the VAMP (vesicle associated membrane protein)-like family (R.III) (VMP7,

240 Cre04.g214750.t1.1), which functions in clathrin-independent vesicular transport and

241 membrane fusion events necessary for protein transport from early endosomes to late 11

242 endosomes (Advani et al., 1999; Prekeris et al., 1999). NO decreased the transcript

243 abundances of endoplasmic reticulum (ER) Qb-SNARE protein and Sec20-family

244 (SEC20, Cre02.g090650.t1.1) (Fig. 7). In addition, NO impacted vesicular protein

245 trafficking that is responsible for the accurate delivery of proteins to correct

246 subcellular compartments (Rosquete et al., 2018) via tethering of a transport vesicle to

247 its target membrane, which is regulated by two classes of tethering factors, long

248 coiled-coil proteins and multi-subunit tethering complexes (MTCs) (Ravikumar et al.,

249 2017). The transport protein particle (TRAPP) complex is a well-studied MTC in yeast

250 and mammals for the regulation of ER-to-Golgi and Golgi-mediated secretion

251 membrane trafficking (TRAnsport Protein Particle, TRAPPI and TRAPPII) and

252 autophagy (TRAPPIII) processes (Kim et al, 2016; Vukasinovic and Zarsky, 2016).

253 Here, we detected several TRAPP genes in Chlamydomonas, in which the transcript

254 abundances of TRAPP I subunits (TRS20, Cre13.g571600.t1.2; TRS23,

255 Cre02.g077500.t1.2; TRS31, Cre03.g146467.t1.1; TRS33, Cre16.g663650.t1.2; BET3,

256 Cre03.g145607.t1.1; BET5, Cre06.g289700.t1.2) and a TRAPPIII subunit (TRS85,

257 Cre01.g024400.t1.1) were transiently decreased by NO burst (Fig. 8). As a catabolic

258 process for recycling cellular materials, autophagy is also induced by NO burst

259 (Supplemental Table S9C), which was reflected by a decrease in TOR1

260 (Cre09.g400553.t1.1) transcript abundance as well as an increase in the transcript

261 abundance of ATG3 (Cre02.g102350.t1.2), ATG4 (Cre12.g510100.t1.1), ATG5

262 (Cre14.g630907.t1.1), ATG6 (Cre01.g020264.t1.1), ATG7 (Cre03.g165215.t1.1),

263 ATG8 (Cre16.g689650.t1.2), and ATG9 (Cre09.g391500.t1.1) (Fig. 9). However, NO

264 decreased the transcript abundance of ATG12 (Cre12.g557000.t1.2) (Fig. 9I). ATG8

265 and ATG8-PE (phosphatidylethanolamine) proteins, which were detected using

266 western blot were also increased by NO burst. The changes in the expression of these

267 genes by SNAP were inhibited in the presence of cPTIO (Fig. 9J). 12

268 NO also modulates protein folding via the transcriptional control of heat shock

269 proteins (HSPs) that control protein folding and homeostasis in Chlamydomonas

270 (Schroda et al., 2015). An overview of how strongly NO treatment impacts expression

271 levels for each HSP gene, including DNAJ-like proteins, small HSPs, HSP60s,

272 HSP70s, HSP90s, and HSP100s, is provided in Supplemental Table S9D. HSP70D

273 (Cre12.g535700.t1.1) was significantly decreased by NO burst and CLBP3

274 (Cre02.g090850.t1.1, belonging to the HSP100 family) and small HSPs (HSP22A,

275 Cre07.g318800.t1.2; HSP22C, Cre03.g145787.t1.1; HSP22E, Cre14.g617450.t1.2;

276 HSP22F, Cre14.g617400.t1.2) were significantly increased by NO burst (Fig. 9). The

277 effect of SNAP on the expression of HSPs was inhibited in the presence of cPTIO.

278

279 Induction of the Antioxidant Defense System by NO

280 Through enzymatic and non-enzymatic routes, the antioxidant defense system is

281 induced by NO treatment (Supplemental Table S10). Superoxide dismutase (SOD),

.- 282 responsible for the dismutaton of O2 to H2O2, is among the most important

283 antioxidant defense enzymes in plants (Alscher et al., 2002). Six SODs, FSD1

284 (Cre02.g096150.t1.2), MSD1 (Cre02.g096150.t1.2), MSD2 (Cre13.g605150.t1.2),

285 MSD3 (Cre16.g676150.t1.2), MSD4 (Cre12.g490300.t1.2), and MSD5

286 (Cre17.g703176.t1.1), were detected to have the most abundant expression for FSD1

287 (Supplemental Table S10), in which FSD1 and MSD3 transcript abundances were

288 markedly increased by NO treatment (Fig. 10A-F). Furthermore, the

289 ascorbate-glutathione cycle (AGC), an important defense pathway for detoxification of

290 H2O2, plays a role in the defense of photo-oxidative stress in Chlamydomonas (Barth

291 et al., 2014; Lin et al., 2016, 2018; Chang et al., 2017; Yeh et al., 2019; Kuo et al.,

292 2020b) and was also induced by NO. NO increased the transcript abundance of APX1

293 (Cre02.g087700.t1.2), DHAR1 (Cre10.g456750.t1.2), and GSHR1 13

294 (Cre06.g262100.t1.1), but decreased the transcript abundance of APX2

295 (Cre06.g285150.t1.2), APX4 (Cre05.g233900.t1.2), and MDAR1 (Cre17.g712100.t1.1)

296 (Fig. 10 and Supplemental Fig. S7). The transcript abundance of GSHR2

297 (Cre09.g396252.t1.1) was not affected by NO (Supplemental Fig. S7). For glutathione

298 (GSH) and ascorbate (AsA) biosynthesis, the transcript abundance of GSH1

299 (Cre02.g077100.t1.2; Fig. 10J) and VTC2 (GDP‐L‐galactose phosphorylase,

300 Cre13.g588150.t1.2; Fig. 10K), a key enzyme for AsA biosynthesis (Urzica et al.,

301 2012), increased under NO treatment, while that of GSH2 (Cre17.g708800.t1.1) was

302 not affected (Supplemental Fig. S7E). However, the activity of SOD (Fig. 10L), APX

303 (Fig. 10M), and MDAR (Fig. 10P) was not affected by NO, whereas GR (Fig. 10Q),

304 DHAR (Fig. 10N), and protein (Fig. 10O) did show an increase in activity. These gene

305 expression changes under SNAP treatment were inhibited in the presence of cPTIO

306 (Fig. 10).

307 AsA and GSH homeostasis and their redox states (AsA/dehydroascorbate (DHA)

308 and GSH/GSSG (oxidized glutathione)) were affected in the NO treatment. Total AsA

309 (Fig. 10R) and DHA (Fig. 10T) concentrations increased 1 h after NO treatment and

310 AsA concentration was not affected (Fig. 10S). The AsA redox state decreased after 1

311 h of NO treatment and then recovered (Fig. 10U). Total GSH concentration did not

312 change in the NO treatment (Fig. 10V), while the GSH concentration decreased (Fig.

313 10W) and the GSSG concentration increased (Fig. 10X), which in turn caused a

314 decrease in the GSH redox state that was restored after 3 h (Fig. 10Y). These changes

315 were inhibited in the presence of cPTIO.

316 Furthermore, the transcript abundances of a gene homologous to glutathione

317 peroxidase (GPX5, Cre10.g458450.t1.3) and σ-class glutathione-S-transferase genes

318 (GSTS1, Cre16.g688550.t1.2; GSTS2, Cre01.g062900.t1.3), associated with oxidative

319 stress acclimation and detoxification response in Chlamydomonas (Ledford et al., 14

320 2007; Fischer et al., 2012), also increased under NO treatment. GPX1

321 (Cre02.g078300.t1.1), GPX (Cre08.g358525.t1.1), GPX3 (Cre03.g197750.t1.2), and

322 GPX4 (Cre10.g440850.t1.1) showed a decrease in transcript abundance in the NO

323 treatment (Supplemental Fig. S8).

324 NO regulates the expression of methionine sulfoxide reductase (MSR), which

325 functions in the reversibility of the oxidization of methionine to methionine and the

326 control of redox homeostasis through modulating the redox status of methionine in

327 proteins (Branlant, 2012; Rey and Tarrago 2018). Here, transcript abundances of

328 MSRA3 (Cre08.g380300.t1.1), MSRA5 (Cre10.g464850.t1.1), and MSRB2.2

329 (Cre14.g615100.t1.2) increased under NO treatment while those of MSRA2

330 (Cre06.g257650.t1.2), MSRA4 (Cre01.g012150.t1.2), and MSRB2.1

331 (Cre14.g615000.t1.1) showed a decrease. The transcript abundance of MSRA1

332 (Cre13.g570900.t1.1) was not affected by NO treatment (Supplemental Fig. S8). The

333 presence of cPTIO inhibited the changes of MSR gene expression.

334 335

336 DISCUSSION

337 NO is a cellular messenger that mediates diverse signaling pathways and plays a

338 role in many physiological processes in plants (Lamattina and Polacco, 2007;

339 Besson-Bard et al., 2008). Studying NO burst over a short-term period provided us a

340 chance to elucidate the metabolic shift to a brief NO attack and the following

341 acclimation processes in Chlamydomonas cells. We recently discovered that NO

342 interacts with reactive oxygen species (ROS) to induce cell death in association with

343 autophagy in Chlamydomonas cells under high intensity illumination (Kuo et al.,

344 2020a). Fortunately, ROS over-production and oxidative damage were not found in the

345 0.3 mM SNAP treatment. Thus, the interference of ROS in the short-term response to

346 NO can be excluded. Moreover, the cells collected from the early exponential phase

347 are not nutrient-starved. The role of NO as a factor responsible for SNAP-induced

348 changes was confirmed with the cPTIO treatment, which allowed for a better

349 understanding of the novel components of gene networks in Chlamydomonas cells

350 against sub-lethal NO stress using transcriptome and physiological analyses. After 1 h

351 of NO treatment, a decrease in the expression of genes associated with transcriptional

352 and translational activity reflected an inhibition of metabolism in Chlamydomonas

353 cells under NO stress. However, Chlamydomonas growth was not impacted by

354 sub-lethal NO stress. The present data suggest that the acclimation of Chlamydomonas

355 cells to NO burst can be accomplished by specific metabolic pathways.

356

357 Induction of the NO Scavenging System Allows for the Acclimation of

358 Chlamydomonas to NO Stress

359 A transient upregulation of truncated hemoglobin (THB1, Cre14.g615400.t1.2;

360 THB2, Cre14.g615350.t1.2), flavodiiron proteins (FLVB, Cre16.g691800.t1.1), and

361 cytochrome P450 (CYP55B1, Cre01.g007950.t1.1; Supplemental Table S6 and Fig. 12) 16

362 was found during the period of NO burst. In Chlamydomonas, THB1 with

363 dioxygenase activity that converts NO into nitrate (Calatrava et al., 2017) is

364 upregulated by NO (Sanz-Luque et al., 2015b). Sanz-Luque et al. (2015a) showed that

365 treatment with 0.1 mM DEANONOate, a NO donor, increased THB1 transcript

366 abundance in the NIT2 (the nitrate assimilation-specific regulatory gene) wild type

367 and the nit2 mutant, while THB2 transcript abundance decreased in the NIT2 wild

368 type and showed a slight increase in the nit2 mutant. Chlamydomonas is able to reduce

369 NO to N2O via FLVB under light conditions (Chaux et al., 2017) or CYP55B1 in the

370 dark (Burlacot et al., 2019). Accordingly, the present data indicate that the NO

371 scavenging system is induced by NO burst to prevent a toxic NO effect, thus allowing

372 the implementation of acclimation processes post NO exposure.

373

374 NO Alters Nitrogen/Sulfur Assimilation and Primary Amino Acid Biosynthesis

375 The availability of nitrogen and sulfur are restricted by sudden NO challenge, as

376 reflected by a transient drop in the expression of ammonium transporters and

377 assimilation enzymes, whereas an increased SAC3 expression was observed, which is

378 related to the inhibition of the expression of most ARSs. Moreover, the temporal

379 upregulation of genes involved in the degradation of several amino acids, including

380 sulfur-containing amino acids, under NO stress suggests that NO burst decreased the

381 synthesis of proteins and other nitrogen-containing compounds that use these amino

382 acids as building blocks. NO also causes a reversible inhibition of high-affinity

383 nitrate/nitrite and ammonium transport and NR activity through post-translational

384 regulation (Sanz-Luque et al., 2013). However, time-course changes in transcription

385 levels reveal that the impedance in nitrogen/amino acid and sulfur utilization by NO

386 burst is relieved after 3 h. The upregulation of sulfate transporters (SLT1, SLT2, and

387 SULP3) and GDH1 functioning in the incorporation of ammonium in Chlamydomonas 17

388 (Muñoz-Blanco and Cárdenas, 1989) serves a purpose for improving sulfur

389 availability and amino acid synthesis under NO stress. However, the process by which

390 nitrogen uptake is limited under NO stress is not clear.

391

392 Mechanisms to Overcome NO-Induced Protein Stress

393 A decrease in the expression of the genes encoding the oligosaccharyltransferase

394 complex, including RNP1 (oligosaccharyltransferase complex subunit delta

395 (ribophorin II)), GTR17 (Glycosyltransferase), GTR22, GTR25

396 (oligsaccharyltransferase STT3 subunit), STT3B

397 (dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit STT3B),

398 CANX (calnexin (CANX)), and CRT2 (Calreticulin 2, calcium-binding protein), by

399 NO treatment (Supplemental Table S6) reflects the dysfunction of proteins caused by a

400 blockage of glycosylation in the ER. However, the expression of DAD1, which

401 functions in N-linked glycosylation in the ER (Kelleher and Gilmore, 1994), increased

402 in the NO treatment. The NO-induced downregulation of TRAPPI and III proteins,

403 which are related to the transport of proteins to correct compartments and the

404 formation of autophagosomes (Garcia et al., 2019; Rosquete et al., 2019), also

405 suggests that there was transient transfer of misfolded and damage proteins during NO

406 treatment. Furthermore, the downregulation of SUMO E2 conjugase, UBC9, by NO

407 reflects the inhibition of SUMOylation for post-translational modification of the

408 proteins, which is crucial for the growth of Chlamydomonas cells (Knobbe et al.,

409 2015). Clearly, NO burst causes protein stress in Chlamydomonas.

410 To overcome disorders caused by potentially misfolded proteins, the membrane

411 trafficking system is induced for the degradation of damaged proteins in the lysosome

412 and/or vacuole via autophagy, which was reflected by a transient increase in the

413 expression of E2 and E3 ubiquitin ligases, ESCRT subunits (VSP), SNAREs, and 18

414 ATGs by NO treatment. The increase in the expression of SYNTAXIN-8B-RELATED

415 and VAMP7, which are endosomal syntaxins that mediate the steps of endosomal

416 protein trafﬁcking (Prekeris et al., 1999), in Chlamydomonas cells suggests that there

417 was fusion between autophagosomes (late endosomes) and lysosomes under NO stress.

418 The TEM images show the formation and fusion of large vesicles at 1 h, possibly

419 acting to enclose the cytosolic components for degradation (Xie and Klionsky, 2007;

420 Nakatogawa et al., 2009); the large vesicles were not visible after 6 h (Supplemental

421 Fig. S9). Using autophagy as a lysosome-mediated pathway for the degradation of

422 cytosolic proteins and organelles (Choi et al., 2013), ATG7 is involved in the two

423 ubiquitin-like systems necessary for the selective cytoplasm-to-vacuole targeting (Cvt)

424 pathway and autophagy by activation of ATG12 and ATG8 and assigns them to E2

425 enzymes, ATG10 and ATG3, respectively. During this process, ATG8 conjugates PE to

426 form lipidated ATG8 (ATG8-PE), an autophagosome membrane component and

427 binding partner for autophagy receptors, which act to recruit cargo to lysosomes for

428 catabolism in ubiquitin-dependent and independent manners for selective autophagy.

429 Although ATG12 and VTC1 (vacuolar transport chaperone-like protein,

430 Cre12.g510250 t1.2) were downregulated by NO burst, an increase in the expression

431 of other ATG genes and abundances of ATG/ATG8-PE proteins in NO-treated

432 Chlamydomonas cells demonstrates the induction of autophagy by working with the

433 sorting protein factors involved in the membrane trafficking system for protein

434 degradation and recycling under NO stress. Because the inhibition of TOR activity can

435 trigger autophagy, as evidenced by the increased ATG8 and ATG8-PE proteins in

436 Chlamydomonas (Pérez-Pérez et al., 2010), the decreased expression of TOR1 due to

437 NO treatment indicates that TOR plays a role in autophagy induction. In addition to

438 membrane trafficking, protein chaperone systems (HSP22A, HSP22C, HSP22E,

439 HSP22F, and CLBP6) are induced in Chlamydomonas cells under NO stress. Thus, the 19

440 protein trafficking system (ubiquitination, SNARE, autophagy) and HSPs are evoked

441 as a way to remove aberrantly folded or damaged proteins, allowing Chlamydomonas

442 cells to maintain normal functions under NO stress.

443

444 NO Inhibits Photosynthesis but Induces the Antioxidant Defense System for the

445 Prevention of Oxidative Stress upon Exposure to NO Burst

446 A decrease in the expression of genes encoding proteins related to photosynthesis

447 and photosynthetic activity in Chlamydomonas by NO illustrates the NO-mediated

448 downregulation of PSII activity and the evolutionary rate of photosynthetic O2 at the

449 transcriptional level. Because of the suppression of PSII activity and the

450 photosynthetic O2 evolution rate by the NO treatment at the protein level in higher

451 plants (Takahashi and Yamasaki, 2002; Singh et al., 2007; Wodala et al., 2008; Ördög

452 et al., 2013), the effects of NO on photosynthesis by protein modification cannot be

453 ignored. NO is also a factor leading to the degradation of the cytochrome b6f complex

454 and Rubisco through FtsH and Clp proteases in sulfur (de Mia et al., 2019) or nitrogen

455 (Wei et al., 2014) starved Chlamydomonas cells. However, it is not clear whether these

456 two protein complexes are degraded under NO burst in nutrient sufficient conditions.

457 We did find that the transcript abundances of the FtsH-like proteases FHL4

458 (Cre13.g568400.t1.2) and FHL6 (Cre03.g201100.t1.2), and DEG11

459 (Cre12.g498500.t1.2) were increased by NO burst, whereas the transcript abundances

460 of the ClpP proteases ClpP4 (Cre12.g500950.t1.2) and ClpP5 (Cre12.g486100.t1.2),

461 and the non-catalytic subunit of the ClpP complex, ClpR2 (Cre16.g682900.t1.2)

462 decreased (Supplemental Table S6). However, the role of FtsH-like and DEG proteases

463 responsible for chloroplast protein degradation by NO burst need to be identified.

464 Because the inhibition of photosynthesis is considered the mechanism for algae to

465 acclimate to nitrogen or sulfur limitation to avoid photo-damage (Peltier and Schmidt, 20

466 1991; Grossman, 2000; Saloman et al., 2012), our findings suggest that the transient

467 inhibition of photosynthesis can prevent Chlamydomonas cells from over-producing

468 ROS, which may occur during NO burst. ROS scavenging ability was enhanced with

469 the increased concentration of AsA due to the upregulation of VTC2 and its

470 regeneration rate, owing to the enhanced DHAR expression (transcript abundance,

471 protein, enzyme activity). In addition, the enhanced GSH regeneration rate, supported

472 by the increase in the GR activity and GSHR1 expression as well as increased GSTS1,

473 GSTS2, and GPX5 expression, prevented the over-production of ROS. Tocopherols

474 (Havaur et al., 2005; Krieger-Liszkay and Trebst, 2006) and carotenoids (Ramel et al.,

1 475 2012) are also O2 quenchers, but their concentrations and the transcript abundances of

476 their biosynthetic enzymes decreased in the NO treatment (Supplemental Fig. S10).

1 477 Instead, an increase in the concentration of AsA, an O2 quencher (Kramarenko et al.,

1 478 2006), was responsible for O2 scavenging under NO stress. NO also triggers MSR

479 expression. Although the redox of AsA and GSH as well as the thiol-based redox

480 regulation proteins, TRX2 (thioredoxin-like protein, Cre03.g157800 t1.1), PRX1

481 (2-cys peroxiredoxin, chloroplastic, Cre06.g257601 t1.2), and PRX2 (2-cys

482 peroxiredoxin, Cre02.g114600 t1.2; Supplemental Table S6), were initially decreased

483 by NO burst, they recovered 3 h after treatment (the restoration of TRX2, PRX1, and

484 PRX2 transcript abundance is not shown). Thus, the antioxidant defense system and

485 MSR worked together to prevent oxidative stress and the abrupt loss in cellular redox

486 homeostasis occurring under NO stress.

487

488 Burst Oxidase-Like 2-Dependent Regulation of the Expression of Selective

489 NO-Inducible Genes for Coping with NO Stress

490 NADPH oxidase acts as an important molecular hub during ROS-mediated

491 signaling in plants (Marino et al., 2011) and in the interplay of ROS and NO signaling 21

492 pathways for the regulation of plant metabolism (Suzuki et al., 2011). A previous study

493 has shown that NADPH oxidase is regulated by transcriptional and enzyme activity

494 levels in higher plants (Hu et al., 2020). The NO-mediated suppression of NADPH

495 oxidase activity is due to post-translational modification known as S-nitrosylation

496 (Wang et al., 2013). The analysis of cis-regulatory elements in the gene promoter

497 region of NADPH oxidase showed diverse expression patterns under different

498 conditions (Wang et al., 2013; Kaur and Pati, 2016). In this study, we did not examine

499 whether the S-nitrosylation of NADPH oxidase occurs in Chlamydomonas upon

500 exposure to NO; however, our present data did detect the constant expression of the

501 gene encoding respiratory burst oxidase-like 1 (RBOL1, Cre03.g188300.t1.1), an

502 NADPH oxidase gene (Fig. 12A). In addition, we observed the significant

503 upregulation of RBOL2 (Cre03.g188400.t1.1) (Fig. 12B) in Chlamydomonas after 1 h

504 of NO exposure, followed by the subsequent restoration to a baseline level after 3 h.

505 The activity of NADPH oxidase also increased after exposure to NO (Fig. 12C).

506 Therefore, it is interesting for us to examine whether NADPH oxidase mediates the

507 NO-induced changes in the expression of genes in Chlamydomonas using the rbol2

508 mutant (Fig. 13A,B). Compared to the wild type, the rbol2 mutant had a 66% decrease

509 in NADPH oxidase activity with a low RBOL2 transcript abundance (Fig. 13C),

510 indicating a differential regulation pattern on the expression of the genes upon

511 exposure to NO (0.3 mM SNAP). In response to NO treatment (0.3 mM SNAP), the

512 transcript abundance of RBOL1 (Fig. 13Z) was not affected in the rbol2 mutant and

513 the induction of RBOL2 expression in wild type (CC-5325) was not observed in the

514 rbol2 mutant (Fig. 13AB). In addition, the NO-induced increase in the expression of

515 THB2 (Fig. 13D), CDO1 (Fig. 13E), CDO2 (Fig. 13F), HSP22A (Fig. 13G), HSP22C

516 (Fig. 13H), ATG4 (Fig. 13J), ATG8 (Fig. 13K), LHCBM9 (Fig. 13L), GUN4 (Fig.

517 13M), APX1 (Fig. 13P), GSHR1 (Fig. 13R), GSTS1 (Fig. 13T), GSTS2 (Fig. 13U), 22

518 DHAR1 (Fig. 13W), and PDS1 (Fig. 13Y) in the wild type was not observed in the

519 rbol2 mutant. In contrast to the NO-mediated inhibition of TOR1 (Fig. 13I) and CYC4

520 (Fig. 13N) expression in the wild type, the expression of TOR1 and CYC4 showed a

521 significant increase in the rbol2 mutant after NO treatment. The expression pattern of

522 RBCS1 (Fig. 13P), VTC2 (Fig. 13R), GSH1 (Fig. 13S), MDAR1 (Fig. 13V), and

523 MDAR5 (Fig. 13X) in the rbol2 mutant in the NO treatment was similar to that in the

524 wild type. Compared to the wild type, the NADPH oxidase activity in the rbol2 mutant

525 did not increase after SNAP treatment (Fig. 14A). The rbol2 mutant showed a higher

526 sensitivity to NO stress than the wild type, as reflected by a decrease in rbol2 mutant

527 viability (Fig. 14B) accompanied with bleaching (Fig. 14C) as the SNAP

528 concentration increased ≥ 0.3 mM. Overall, NADPH oxidase (RBOL2) is associated

529 with the expression of the genes encoding proteins for NO scavenging, antioxidant

530 defense system, autophagy, and heat shock proteins that help to resist NO stress in

531 Chlamydomonas.

532

533 CONCLUSIONS

534 The acclimation of Chlamydomonas to sub-lethal NO stress comprises a temporally

535 orchestrated implementation of metabolic processes including 1. a decrease in the NO

536 amount due to scavenging elements, 2. acclimation to nitrogen and sulfur starvation, 3.

537 upregulation of the protein trafficking system (ubiquitin, SNARE, and autophagy) and

538 molecular chaperone system for dynamic regulation of protein homeostasis and

539 function, and 4. transient inhibition of photosynthesis together with the induction of

540 the antioxidant defense system and modification of the redox state to prevent oxidative

541 stress. NADPH oxidase (RBOL2)-dependent molecular events underlie part of the

542 acclimation mechanisms in Chlamydomonas related to coping with NO stress. The

543 direct targets of NO and the mechanisms for the transcriptional regulation of RBOL2 23

544 in Chlamydomonas are needed to be clarified in the future.

545

546 MATERIALS AND METHODS

547 Algal Culture and SNAP Treatments

548 The green alga Chlamydomonas reinhardtii, strain CC-125 (mt-), was obtained from

549 the Chlamydomonas Resource Center (USA) and photoheterotrophically cultured in

550 Tris-acetate phosphate medium (TAP) (Harris, 1989) with a trace element solution in

551 125 mL flasks (PYREX, Germany) and agitated on an orbital shaking incubator

552 (model OS701, TKS company, Taipei, Taiwan) (150 rpm) under continuous

553 illumination with white light (50 μmol·m-2·s-1) at 25°C. For chemical treatments, 50

554 mL cultures were grown to a cell density of 3–5×106 cells·mL-1, and after

555 centrifugation at 1,600 ×g for 3 min, the supernatant was discarded. The pellet was

556 suspended in fresh TAP medium and centrifuged again. Then, the pellet was

557 re-suspended in fresh TAP medium to a cell density of 3×106 cells·mL-1. Ten milliliters

558 of culture was transferred to a 100-mL beaker (internal diameter: 3.5 cm) for

559 pre-incubation at 25°C in 50 μmol·m-2·s-1 conditions for 1.5 h in an orbital shaker

560 (model OS701, TKS company, Taipei, Taiwan) at a speed of 150 rpm. Then, the algal

561 cells were subjected to treatments at 25°C. SNAP was treated in different

562 concentrations from 0.1, 0.3, to 1.0 mM in the presence or absence of 0.4 mM cPTIO.

563 DMSO was used as the control because SNAP was dissolved in DMSO. Each

564 treatment included three biological independent replicates (n=3). For the determination

565 of cell growth and the estimation of several physiological and biochemical parameters,

566 the number of cells in a 1-mL sample was counted using a hemocytometer. Samples

567 taken before (0 min) and after treatment were centrifuged at 5,000 ×g for 5 min, and

568 the pellet was fixed in liquid nitrogen and stored in a -70°C freezer until analysis.

569

570 Detection of NO Production

571 An NO-sensitive fluorescent dye, DAF-FM diacetate (Invitrogen Life Technologies,

572 Carlsbad, CA, USA) (Kojima et al., 1998), was used to measure NO production

573 following our previous studies. DAF-FM diacetate is a pH-insensitive fluorescent dye

574 that emits fluorescence after reaction with an active intermediate of NO (Kojima et al.,

575 1998; Chang et al., 2013). The cells were pre-incubated in TAP medium containing 5

576 μM DAF-FM diacetate for 60 min at 25°C under 50 μmol·m-2·s-1 conditions, then

577 washed twice with fresh TAP medium, and transferred to 50 μmol·m-2·s-1 for chemical

578 treatment. The fluorescence was detected via fluorescence microscopy and

579 spectrophotometry.

580 581 Determination of Chlorophyll a Fluorescence

582 Chlorophyll a fluorescence parameters were employed to determine the activity of

583 PSII using an AP-C 100 (AquaPen, Photon Systems Instruments, Brno, Czech

584 Republic). A 0.5-mL aliquot of an algal culture was diluted with TAP medium to an

-1 585 OD750 = 0.10–0.15 with a chlorophyll a content of 1.5–2.2 μg·mL . A 2-mL aliquot of

586 diluted algal cells was then transferred to an AquaPen cuvette and subjected to a pulse

587 of saturating light of 4,000 μmol·m-2·s-1 PAR to obtain the light-adapted minimal

588 fluorescence (Ft) and the light-adapted maximal fluorescence (Fm'). To determine the

589 maximum PSII activity, Fv/Fm (=Fm - Fo/Fm), 2 mL of diluted algal cells in an

590 AquaPen cuvette was incubated in the dark for 20 min and then flushed with saturated

-2 -1 591 light (4,000 μmol photons·m ·s ) to obtain the dark-adapted minimal fluorescence (Fo)

592 and dark-adapted maximal fluorescence (Fm). The active PSII activity, Fv'/Fm' = Fm' -

593 Ft/Fm', and the maximum PSII activity, Fv/Fm = Fm– Fo/Fm, were then calculated.

594 The rapid induction of chlorophyll a fluorescence was also determined via the OJIP

595 test (Strasser and Strasser, 1995; Strasser et al., 2000). The inflections in the O-J-I-P

596 curve represent the heterogeneity of the process during photochemical action; peak J

- - - - 597 represents the momentary maximum level of QA , QA QB, and QA QB ; peak I

- 2- - 2- 598 represents the level of QA QB ; and peak P represents the maximum level of QA , QB ,

599 and PQH2 (Stirbet et al., 1998). The fluorescence values at time intervals

600 corresponding to the O-J-I-P peaks were recorded as follows: Fo = fluorescence

601 intensity at 50 µs; FJ = fluorescence intensity at peak J (at 2 ms); Fi = fluorescence

602 intensity at peak I (at 60 ms); Fm = maximal fluorescence intensity at the peak P; Fv =

603 Fm - Fo (maximal variable fluorescence); Vj = (Fj - Fo)/(Fm - Fo); Vi = (Fi - F0)/(Fm -

604 Fo); and Fm/Fo; Fv/Fo; Fv/Fm.

605

606 Enzyme Assay and Determination of AsA and GSH

607 SOD activity was assayed according to (Yashida et al., 2003). GR and APX activity

608 was determined according to the methods described by Lin et al. (2018) and Kuo et al.

609 (2020b), respectively. Protein concentrations were quantified using the Coomassie

610 Blue dye binding method (Bradford, 1976) using a concentrated dye purchased from

611 BioRad (500-0006, Hercules, CA, USA). AsA and DHA concentrations were

612 determined by an ascorbate oxidase based method according to Lin et al. (2016). GSH

613 and oxidized glutathione (GSSG) were extracted from the frozen algal cell pellet

614 (obtained from 5 mL samples) using 5% (w/v) trichloroacetic acid (TCA) and

615 determined at 412 nm according to Lin et al. (2018).

616

617 Determination of NADPH oxidase activity

618 NADPH oxidase activity was determined according to Kaundal et al.’s (2012)

-. 619 method based on O2 production. After homogenization, centrifugation at 10,000 ×g,

620 and then ultra-centrifugation at 203,000 ×g for collection of total membranes, the

621 pellet was suspended in 1 mL ice-cold 10 mM Tris-HCl (pH 7.4). The reaction mixture

622 consisted of 50 mM Tris-HCl buffer (pH 7.5), 1 mM XTT

623 (3-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfoni

624 c acid hydrate), 1 mM NADPH, and 5 μg membrane protein. The reduction rate of

-. 625 XTT by O2 detected at 492 nm was used to calculate enzyme activity; the extinction

626 coefficient used was 2.16 × 104 M-1 cm-1.

627

628 RNA Isolation, cDNA Synthesis, Transcriptomic Analysis, and mRNA

629 Quantification via Real-Time Quantitative PCR

630 Total RNA was extracted using the TriPure Isolation Reagent (Roche Applied

631 Science, Mannheim, Germany) according to the manufacturer’s instructions. The

632 methods for cDNA library preparation, Illumina sequencing, and sequence analysis are

633 described in Supplemental Fig. S6. For qPCR assay, the total RNA concentration was

634 adjusted to 2.95 μg total RNA·μL-1 and treated with DNase (TURBO DNA-freeTM Kit,

635 Ambion Inc., The RNA Company, USA) to remove residual DNA. Then 1.5 μg of

636 total RNA was used for the preparation of cDNA. cDNA was amplified from the

637 poly-(A+) tail using Oligo (dT)12-18 with the VersoTM cDNA Kit (Thermo Fisher

638 Scientific Inc., Waltham, MA, USA), and the volume was adjusted to a concentration

639 of 30 ng·mL-1 based on original RNA quantity in each sample. The primers for the

640 targeted genes are listed in Supplemental Table S11. The real-time quantitative PCR

641 was performed using a LightCycler 480 system (Roche Applied Science, Mannheim,

642 Germany). A PCR master mix was prepared with the LightCycler 480 SYBR Green I

643 Master Kit (Roche Applied Science, Mannheim, Germany). Each reaction was

644 performed in a total volume of 10 μL, containing 1× LightCycler 480 SYBR Green I

645 Master Mix, the selected concentration of each primer, and cDNA corresponding to 30

646 or 50 ng·μL-1 RNA in the reverse transcriptase reaction. The amplification program

647 consisted of an initial denaturation at 95°C for 5 min, followed by 50 amplification

648 cycles of annealing at 60°C for 10 s, elongation at 72°C for 5 s, real-time fluorescence

649 measurements, and finally, denaturation at 95°C for 15 s. The 2-∆∆CT method was used

650 to calculate the relative change in mRNA level normalized to a reference gene (UBC,

651 NCBI: AY062935) and the fold increase was calculated relative to the control RNA

652 sample at 0 min. Because the results based on the EF-1α internal control were similar

653 to those based on UBC, the relative changes in the levels of gene transcription were

654 expressed based on UBC.

655

656 Western Blots

657 Soluble protein was extracted according to Kuo et al. (2020a). For each sample, 30

658 μg of protein was loaded into each lane, resolved on a 15% SDS-PAGE gel, and

659 transferred to a polyvinylidene fluoride membrane for antibody binding with rabbit

660 polyclonal anti-ATG8 antibody (ab77003; Abcam, Cambridge, UK), anti-CrDHAR

661 (LKT BioLaboratories Ltd., Taoyuan, Taiwan), or a mouse monoclonal antibody

662 against α-tubulin (ab11304; Abcam, Cambridge, UK). Following the incubation with

663 horseradish peroxidase-conjugated secondary antibodies (MD20878; KPL,

664 Gaithersburg, MD, USA), the immunoblots were visualized and the relative abundance

665 of ATG8, ATG8-PE, or CrDHAR1 protein was estimated based on α-tubulin intensity.

666

667 Statistics

668 Three independent biological replicates were performed and all experiments were

669 repeated at least three times. Because the replications showed similar trends, only 28

670 the results from one replicate were shown in this paper. Statistical analyses were

671 performed using SPSS (SPSS 15.0, Chicago, IL, USA). Significant differences

672 between means were analyzed using Student’s t-test or Scheffe’s test following

673 significant analysis of variance for the controls and treatments at P < 0.05.

674

675 Accession Numbers

676 The transcriptome sequences can be accessed from the Sequence Read Archive

677 (SRA) website using the BioProject accession number: PRJNA629395

678 (https://www.ncbi.nlm.nih.gov/books/NBK158898/) and the BioSample accessions

679 SAMN14775313, SAMN14775314, SAMN14775315, SAMN14775316,

680 SAMN14775317, SAMN14775318, SAMN14775319, SAMN14775320,

681 SAMN14775321, and SAMN14775322.

682

683 Supplemental Data

684 The following supplemental materials are available:

685 Supplemental Tables S1–S11.

686 Supplemental Figures S1–S10.

687

688 ACKNOWLEDGEMENTS

689 We thank Dr. Su-Chiang Fang, Academia Sinica Biotechnology Center in Southern

690 Taiwan, for assistance with culturing Chlamydomonas and manuscript preparation.

691

692

693 Figure Legends

694 Figure 1. Microscopic observation (A) and spectrophotometric determination (B) of

695 nitric oxide fluorescence (DAF-FM), cell viability (C), cell growth (D), and 29

696 appearance (E) in Chlamydomonas reinhardtii in response to 0.1, 0.3, or 1.0 mM

697 SNAP in the presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ±

698 SD (n = 3). Different symbols indicate significant differences between treatments

699 (Scheffe’s test, P < 0.05).

700

701 Figure 2. The active (Fv′/Fm′) (A) and maximum (Fv/Fm) (B) PSII activity,

702 photosynthetic O2 evolution rate (C), and respiration rate (D) in C. reinhardtii after

703 exposure to 0.1, 0.3, or 1.0 mM SNAP in the presence or absence of 0.4 mM cPTIO.

704 Data are expressed as the mean ± SD (n = 3). Different symbols indicate significant

705 differences between treatments (Scheffe’s test, P < 0.05).

706

707 Figure 3. Time-course changes in the transcript abundances of PSBP4 (A), LHCBM9

708 (B), Lhl1 (C), Lhl2 (D), Lhl3 (E), Lhl4 (F), PCY1 (G), CYC4 (H), CHLH (I), CHLI1

709 (J), CHLI2 (K), CHLD (L), and GUN4 (M) in C. reinhardtii upon exposure to 0.3 mM

710 SNAP in the presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ±

711 SD (n = 3). Different symbols indicate significant differences between treatments

712 (Scheffe’s test, P < 0.05).

713

714 Figure 4. Time-course changes in the transcript abundances of RBCS1 (A), RPE2 (B),

715 PGK1 (C), GAP4 (D), TPI1 (E), SBP2 (F), PPR2 (G), RCA2 (H), CP12 (I), RMT1 (J),

716 RMT2 (K), and PGP3 (L) in C. reinhardtii after exposure to 0.3 mM SNAP in the

717 presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ± SD (n = 3).

718 Different symbols indicate significant differences between treatments (Scheffe’s test, P

719 < 0.05).

720

721 Figure 5. Time-course changes in the transcript abundances of AMT3 (A), AMT6 (B), 30

722 AMT7 (C), GSN1 (D) GLN1 (E), GDH1 (F), CSD4 (G), CDO1 (H), CDO2 (I),

723 TAUD1 (J), AST5 (K), ATA2 (L), BCKDHA (N), and BCKDHB (O) in C. reinhardtii

724 in response to 0.3 mM SNAP in the presence or absence of 0.4 mM cPTIO. Data are

725 expressed as the mean ± SD (n = 3). Different symbols indicate significant differences

726 between treatments (Scheffe’s test, P < 0.05).

727

728 Figure 6. Time-course changes in the transcript abundances of SAC3 (A), SUOX (B),

729 ARS3 (C), ARS7 (E), ARS5 (D), ARS11 (G), ARS13 (H), ARS14 (I), ARS16 (J),

730 SLT1 (K), SLT2 (L), and SULP3 (M) in C. reinhardtii after exposure to 0.3 mM SNAP

731 in the presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ± SD (n

732 = 3). Different symbols indicate significant differences between treatments (Scheffe’s

733 test, P < 0.05).

734

735 Figure 7. Time-course changes in the transcript abundances of AAA+-type ATPase

736 (A), ubiquitin-protein ligase E2 (B), UBC9 (C), UBC3 (D), UBE3A (E), probable E3

737 ubiquitin-protein ligase HERC1 (F), ubiquitin fusion degradation protein (G), VPS23

738 (H), VPS28 (I), VPS37 (J), VPS2A (K), RAB5 (L), SEC20 (M),

739 SYNTAXIN-8B-RELATED (N), and VAMP (O) in C. reinhardtii after exposure to 0.3

740 mM SNAP in the presence or absence of 0.4 mM cPTIO. Data are expressed as the

741 mean ± SD (n = 3). Different symbols indicate significant differences between

742 treatments (Scheffe’s test, P < 0.05).

743

744 Figure 8. Time-course changes in the transcript abundances of TRS20 (A), TRS23 (B),

745 TRS31 (C), TRS33 (D), TRS85 (E), BET3 (F), and BET5 (G) in C. reinhardtii after

746 exposure to 0.3 mM SNAP in the presence or absence of 0.4 mM cPTIO. Data are

747 expressed as the mean ± SD (n = 3). Different symbols indicate significant differences 31

748 between treatments (Scheffe’s test, P < 0.05).

749

750 Figure 9. Time-course changes in the transcript abundances of autophagy-related

751 genes and heat-shock proteins and the protein abundances of ATG8 in C. reinhardtii

752 upon exposure to 0.3 mM SNAP in the presence or absence of 0.4 mM cPTIO. A.

753 TOR1; B. ATG3; C. ATG4; D. ATG5; E. ATG6; F. ATG7; G. ATG8; H. ATG9; I.

754 ATG12; J. ATG8 (square symbol) and ATG8-PE (circle symbol) protein abundances;

755 K. HSP70D; L. CLBP3; M. HSP22A; N. HSP22C; O. HSP22E; P. HSP22F. Data are

756 expressed as the mean ± SD (n = 3). Different symbols indicate significant differences

757 between treatments (Scheffe’s test, P < 0.05).

758

759 Figure 10. Time-course changes in the transcript abundances of FSD1 (A), MSD1 (B),

760 MSD2 (C), MSD3 (D), MSD4 (E), MSD5 (F), APX1 (G), DHAR1 (H), GSHR1 (I),

761 GSH1 (J), and VTC2 (K); the activity of APX (L), DHAR (M), MDAR (N), and GR

762 (O); the concentrations of total AsA (P), AsA (Q), and DHA (R); the ratio of

763 AsA/DHA (S); the concentrations of total GSH (T), GSH (U), and GSSG (V); and the

764 ratio of GSH/GSSG (W) in C. reinhardtii after exposure to 0.3 mM SNAP in the

765 presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ± SD (n = 3).

766 Different symbols indicate significant differences between treatments (Scheffe’s test, P

767 < 0.05).

768

769 Figure 11. Time-course changes in the transcript abundances of THB1 (A), THB2 (B),

770 FLVB (C), and CYP55B1 (D) in C. reinhardtii after exposure to 0.3 mM SNAP in the

771 presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ± SD (n = 3).

772 Different symbols indicate significant differences between treatments (Scheffde’s test,

773 P < 0.05). 32

774

775 Figure 12. Time-course changes in the transcript abundances of RBOL1 (A) and

776 RBOL2 (B) and the activity of NADPH oxidase (C) in C. reinhardtii after exposure to

777 0.3 mM SNAP in the presence or absence of 0.4 mM cPTIO. Data are expressed as the

778 mean ± SD (n = 3). Different symbols indicate significant differences between

779 treatments (Scheffe’s test, P < 0.05).

780

781 Figure 13. Isolation of the Chlamydomonas rbol2 mutant and its transcript abundances

782 of several genes in response to 0.3 mM SNAP in the presence or absence of 0.4 mM

783 cPTIO. (A) Schematic representation of insertion sites of the APHVIII cassettes in the

784 genomic sequence of RBOL2. Tall boxes denote exons, gray boxes indicate protein

785 coding regions and filled boxes show 5’ and 3’ untranslated regions (UTRs) and the

786 promoter region. Arrows indicate primer locations used to detect APHVIII cassette

787 insertions. (B) Genotyping of the rbol2 mutant. Genomic DNA fragments were

788 amplified by PCR using the primer sets indicated in (A). (C) The rbol2 mutant showed

789 low RBOL2 transcript abundance and NADPH oxidase activity but a normal RBOL1

790 expression compared to the CC-5325 wild type. The data are expressed as the mean ±

791 SD (n = 3) from three independent biological replicates, and * indicates a significant

792 difference between CC-5325 and the rbol2 mutant using t-test (P < 0.05). The

793 transcript abundance of THB2 (D), CDO1 (E), CDO2 (F), HSP22A (G), HSP22C (H),

794 TOR1 (I), ATG4 (J), ATG8 (K), LHCBM9 (L), GUN4 (M), CYC4 (N), RBCS1 (O),

795 APX1 (P), VTC2 (Q), GSHR1 (R), GSH1 (S), GSTS1 (T), GSTS2 (U), MDAR1 (V),

796 DHAR1 (W), MSAR5 (X), PDS1 (Y), RBOL1 (Z), and RBOL2 (AB). The data are

797 expressed as the mean ± SD (n = 3), in which different symbols indicate significant

798 differences between treatments ( Scheffe’s test, P < 0.05).

799 33

800 Figure 14. The effects of SNAP treatment on NADPH oxidase activity (A), the

801 viability of Chlamydomonas after 4 days of culture on an agar plate including 6 h

802 SNAP treatment (B) and the appearance of cells 6 h after SNAP treatment. SNAP was

803 applied at 0, 0.1, 0.3, or 1.0 mM concentrations. Data are expressed as the mean ± SD

804 (n = 3) for NADPH oxidase activity. Different symbols indicate significant differences

805 between treatments (Scheffe’s test, P < 0.05).

806

807

2 Respiratory Burst Oxidase-Like 2

3 Eva YuHua Kuo and Tse-Min Lee 4

1 2 Figure 1. Microscopic observation (A) and spectrophotometric determination (B) of 3 nitric oxide fluorescence (DAF-FM), cell viability (C), cell growth (D), and 4 appearance (E) in Chlamydomonas reinhardtii in response to 0.1, 0.3, or 1.0 mM 5 SNAP in the presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ± 6 SD (n = 3). Different symbols indicate significant differences between treatments 7 (Scheffe’s test, P < 0.05).

2 Figure 2. The active (Fv′/Fm′) (A) and maximum (Fv/Fm) (B) PSII activity,

3 photosynthetic O2 evolution rate (C), and respiration rate (D) in C. reinhardtii after 4 exposure to 0.1, 0.3, or 1.0 mM SNAP in the presence or absence of 0.4 mM cPTIO. 5 Data are expressed as the mean ± SD (n = 3). Different symbols indicate significant 6 differences between treatments (Scheffe’s test, P < 0.05).

1 2 Figure 3. Time-course changes in the transcript abundances of PSBP4 (A), LHCBM9 3 (B), Lhl1 (C), Lhl2 (D), Lhl3 (E), Lhl4 (F), PCY1 (G), CYC4 (H), CHLH (I), CHLI1 4 (J), CHLI2 (K), CHLD (L), and GUN4 (M) in C. reinhardtii upon exposure to 0.3 5 mM SNAP in the presence or absence of 0.4 mM cPTIO. Data are expressed as the 6 mean ± SD (n = 3). Different symbols indicate significant differences between 7 treatments (Scheffe’s test, P < 0.05).

1 2 Figure 4. Time-course changes in the transcript abundances of RBCS1 (A), RPE2 (B), 3 PGK1 (C), GAP4 (D), TPI1 (E), SBP2 (F), PPR2 (G), RCA2 (H), CP12 (I), RMT1 (J), 4 RMT2 (K), and PGP3 (L) in C. reinhardtii after exposure to 0.3 mM SNAP in the 5 presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ± SD (n = 3). 6 Different symbols indicate significant differences between treatments (Scheffe’s test, 7 P < 0.05).

1 2 Figure 5. Time-course changes in the transcript abundances of AMT3 (A), AMT6 (B), 3 AMT7 (C), GSN1 (D) GLN1 (E), GDH1 (F), CSD4 (G), CDO1 (H), CDO2 (I), 4 TAUD1 (J), AST5 (K), ATA2 (L), BCKDHA (N), and BCKDHB (O) in C. reinhardtii 5 in response to 0.3 mM SNAP in the presence or absence of 0.4 mM cPTIO. Data are 6 expressed as the mean ± SD (n = 3). Different symbols indicate significant differences 7 between treatments (Scheffe’s test, P < 0.05).

1 2 Figure 6. Time-course changes in the transcript abundances of SAC3 (A), SUOX (B), 3 ARS3 (C), ARS7 (E), ARS5 (D), ARS11 (G), ARS13 (H), ARS14 (I), ARS16 (J), 4 SLT1 (K), SLT2 (L), and SULP3 (M) in C. reinhardtii after exposure to 0.3 mM 5 SNAP in the presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ± 6 SD (n = 3). Different symbols indicate significant differences between treatments 7 (Scheffe’s test, P < 0.05).

1 2 Figure 7. Time-course changes in the transcript abundances of AAA+-type ATPase 3 (A), ubiquitin-protein ligase E2 (B), UBC9 (C), UBC3 (D), UBE3A (E), probable E3 4 ubiquitin-protein ligase HERC1 (F), ubiquitin fusion degradation protein (G), VPS23 5 (H), VPS28 (I), VPS37 (J), VPS2A (K), RAB5 (L), SEC20 (M), 6 SYNTAXIN-8B-RELATED (N), and VAMP (O) in C. reinhardtii after exposure to 7 0.3 mM SNAP in the presence or absence of 0.4 mM cPTIO. Data are expressed as 8 the mean ± SD (n = 3). Different symbols indicate significant differences between 9 treatments (Scheffe’s test, P < 0.05).

1 2 Figure 8. Time-course changes in the transcript abundances of TRS20 (A), TRS23 3 (B), TRS31 (C), TRS33 (D), TRS85 (E), BET3 (F), and BET5 (G) in C. reinhardtii 4 after exposure to 0.3 mM SNAP in the presence or absence of 0.4 mM cPTIO. Data 5 are expressed as the mean ± SD (n = 3). Different symbols indicate significant 6 differences between treatments (Scheffe’s test, P < 0.05).

1 2 Figure 9. Time-course changes in the transcript abundances of autophagy-related 3 genes and heat-shock proteins and the protein abundances of ATG8 in C. reinhardtii 4 upon exposure to 0.3 mM SNAP in the presence or absence of 0.4 mM cPTIO. A. 5 TOR1; B. ATG3; C. ATG4; D. ATG5; E. ATG6; F. ATG7; G. ATG8; H. ATG9; I. 6 ATG12; J. ATG8 (square symbol) and ATG8-PE (circle symbol) protein abundances; 7 K. HSP70D; L. CLBP3; M. HSP22A; N. HSP22C; O. HSP22E; P. HSP22F. Data are 8 expressed as the mean ± SD (n = 3). Different symbols indicate significant differences 9 between treatments (Scheffe’s test, P < 0.05).

2 3 Figure 10. Time-course changes in the transcript abundances of FSD1 (A), MSD1 (B), 4 MSD2 (C), MSD3 (D), MSD4 (E), MSD5 (F), APX1 (G), DHAR1 (H), GSHR1 (I), 5 GSH1 (J), and VTC2 (K); the activity of APX (L), DHAR (M), MDAR (N), and GR 6 (O); the concentrations of total AsA (P), AsA (Q), and DHA (R); the ratio of 7 AsA/DHA (S); the concentrations of total GSH (T), GSH (U), and GSSG (V); and the 8 ratio of GSH/GSSG (W) in C. reinhardtii after exposure to 0.3 mM SNAP in the 9 presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ± SD (n = 3). 10 Different symbols indicate significant differences between treatments (Scheffe’s test, 11 P < 0.05).

1 2 Figure 11. Time-course changes in the transcript abundances of THB1 (A), THB2 (B), 3 FLVB (C), and CYP55B1 (D) in C. reinhardtii after exposure to 0.3 mM SNAP in the 4 presence or absence of 0.4 mM cPTIO. Data are expressed as the mean ± SD (n = 3). 5 Different symbols indicate significant differences between treatments (Scheffde’s test, 6 P < 0.05).

1 2 Figure 12. Time-course changes in the transcript abundances of RBOL1 (A) and 3 RBOL2 (B) and the activity of NADPH oxidase (C) in C. reinhardtii after exposure to 4 0.3 mM SNAP in the presence or absence of 0.4 mM cPTIO. Data are expressed as 5 the mean ± SD (n = 3). Different symbols indicate significant differences between 6 treatments (Scheffe’s test, P < 0.05).

1 2 Figure 13. Isolation of the Chlamydomonas rbol2 mutant and its transcript 3 abundances of several genes in response to 0.3 mM SNAP in the presence or absence 4 of 0.4 mM cPTIO. (A) Schematic representation of insertion sites of the APHVIII 5 cassettes in the genomic sequence of RBOL2. Tall boxes denote exons, gray boxes 6 indicate protein coding regions and filled boxes show 5’ and 3’ untranslated regions

bioRxiv preprint doi: https://doi.org/10.1101/2021.03.30.437739; this version posted March 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 (UTRs) and the promoter region. Arrows indicate primer locations used to detect 2 APHVIII cassette insertions. (B) Genotyping of the rbol2 mutant. Genomic DNA 3 fragments were amplified by PCR using the primer sets indicated in (A). (C) The 4 rbol2 mutant showed low RBOL2 transcript abundance and NADPH oxidase activity 5 but a normal RBOL1 expression compared to the CC-5325 wild type. The data are 6 expressed as the mean ± SD (n = 3) from three independent biological replicates, and 7 * indicates a significant difference between CC-5325 and the rbol2 mutant using t-test 8 (P < 0.05). The transcript abundance of THB2 (D), CDO1 (E), CDO2 (F), HSP22A 9 (G), HSP22C (H), TOR1 (I), ATG4 (J), ATG8 (K), LHCBM9 (L), GUN4 (M), CYC4 10 (N), RBCS1 (O), APX1 (P), VTC2 (Q), GSHR1 (R), GSH1 (S), GSTS1 (T), GSTS2 11 (U), MDAR1 (V), DHAR1 (W), MSAR5 (X), PDS1 (Y), RBOL1 (Z), and RBOL2 12 (AB). The data are expressed as the mean ± SD (n = 3), in which different symbols 13 indicate significant differences between treatments ( Scheffe’s test, P < 0.05).

1 2 Figure 14. The effects of SNAP treatment on NADPH oxidase activity (A), the 3 viability of Chlamydomonas after 4 days of culture on an agar plate including 6 h 4 SNAP treatment (B) and the appearance of cells 6 h after SNAP treatment. SNAP was 5 applied at 0, 0.1, 0.3, or 1.0 mM concentrations. Data are expressed as the mean ± SD 6 (n = 3) for NADPH oxidase activity. Different symbols indicate significant 7 differences between treatments (Scheffe’s test, P < 0.05).

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